Soluble needle arrays for delivery of influenza vaccines

ABSTRACT

Influenza vaccines are administered using solid biodegradable microneedles. The microneedles are lubricated from the influenza vaccine in combination with solid excipient(s) and, after penetrating the skin, they dissolve in situ and release the vaccine to the immune system. The influenza vaccine is (i) a purified influenza virus surface antigen vaccine, rather than a live vaccine or a whole-virus or split inactivated vaccine (ii) an influenza vaccine prepared from viruses grown in cell culture, not eggs, (iii) a monovalent influenza vaccine e.g. for immunising against a pandemic strain, (iv) a bivalent vaccine, (v) a tetravalent or &gt;4 -valent vaccine, (vi) mercury-free vaccine, or (vii) a gelatin-free vaccine.

This application claims the benefit of U.S. provisional application61/401,844 (filed Aug. 20, 2010), the complete contents of which arehereby incorporated herein by reference for all purposes.

TECHNICAL FIELD

This invention is in the field of influenza vaccination.

BACKGROUND ART

Various forms of influenza virus vaccine are currently available (e.g.see chapters 17 & 18 of reference 1) and current vaccines are basedeither on inactivated or live attenuated viruses. Inactivated vaccinesare administered by intramuscular or intradermal injection, whereas livevaccines are administered intranasally.

It is an object of the invention to provide a different way ofadministering inactivated influenza vaccines, and in particular a moreconvenient way e.g. which does not require medical personnel, and whichmay thus be sold in an over-the-counter setting,

DISCLOSURE OF THE INVENTION

According to the invention, influenza vaccines are administered usingsolid biodegradable microneedles. The microneedles are fabricated fromthe influenza vaccine in combination with solid excipient(s) and, afterpenetrating the skin, they dissolve in situ and release the vaccine tothe immune system. In preferred embodiments the influenza vaccine is (i)a purified influenza virus surface antigen vaccine, rather than a livevaccine or a whole-virus or split inactivated vaccine (ii) an influenzavaccine prepared from viruses grown in cell culture, not eggs, (iii) amonovalent influenza vaccine e.g. for immunising against a pandemicstrain, (iv) a bivalent vaccine, (v) a tetravalent or >4-valent vaccine,(vi) a mercury-free vaccine, and/or (vii) a gelatin-free vaccine.

Thus the invention provides a skin patch comprising a plurality of solidbiodegradable microneedles, wherein the microneedles comprise a mixtureof (i) a biosoluble and biodegradable matrix material and (ii) aninfluenza vaccine selected from the group consisting of a purifiedinfluenza virus surface antigen vaccine, an influenza vaccine preparedfrom viruses grown in cell culture, a monovalent influenza vaccine, abivalent vaccine, a tetravalent or >4-valent vaccine, a mercury-freevaccine, and a gelatin-free vaccine. The vaccine can have one or more ofthese features. This patch can be used to deliver an influenza vaccineto a subject via their skin, and so can be used in a method for raisingan immune response in a mammal.

The invention also provides a process for preparing a skin patchcomprising a plurality of solid biodegradable microneedles, comprisingsteps of: (i) mixing a biosoluble and biodegradable matrix material withan influenza vaccine selected from the group consisting of a purifiedinfluenza virus surface antigen vaccine, an influenza vaccine preparedfrom viruses grown in cell culture, a monovalent influenza vaccine, abivalent vaccine, a tetravalent or >4-valent vaccine, a mercury-freevaccine, and a gelatin-free vaccine; and (ii) adding the mixture fromstep (i) to a mold containing cavities for forming microneedles.

The invention also provides an aqueous liquid or solid materialcomprising (i) a biosoluble and biodegradable matrix material and (ii)an influenza vaccine selected from the group consisting of a purifiedinfluenza virus surface antigen vaccine, an influenza vaccine preparedfrom viruses grown in cell culture, a monovalent influenza vaccine, abivalent vaccine, a tetravalent or >4-valent vaccine, a mercury-freevaccine, and a gelatin-free vaccine. This material is suitable forpreparing a patch of the invention.

The invention also provides a skin patch comprising a plurality of solidbiodegradable microneedles, wherein the microneedles comprise a mixtureof (i) a biosoluble and biodegradable matrix material and (ii) aninfluenza virus hemagglutinin, wherein the amount of influenza virushemagglutinin per patch is ≤16 μg per strain. This patch can be used todeliver an inactivated influenza vaccine to a subject via their skin,and so can be used in a method for raising an immune response in amammal.

The invention also provides a process for preparing a skin patchcomprising a plurality of solid biodegradable microneedles, comprisingsteps of: (i) mixing a biosoluble and biodegradable matrix material withan influenza vaccine; and (ii) adding the mixture from step (i) to amold containing cavities for forming microneedles, wherein the amount ofmixture added in step (ii) provides a patch having ≤16 μg influenzavirus hemagglutinin per strain per patch.

The invention also provides an aqueous liquid or solid materialcomprising (i) a biosoluble and biodegradable matrix material and (ii)an influenza virus hernagglutinin at a concentration of ≤16 μg perstrain. This material is suitable for preparing a patch of theinvention.

The invention also provides a process for determining the amount ofinfluenza hemagglutinin in a skin patch, wherein (a) the patch comprisesa biosoluble & biodegradable matrix material and an influenza vaccine,and (b) the process comprises steps of: (i) dissolving the patch in asolvent to provide a dissolved patch solution; and (ii) assayinghemagglutinin in the dissolved patch solution by enzyme-linkedimmunosorbent assay (ELISA).

The invention also provides a process for determining the amount ofinfluenza hemagglutinin in a skin patch, wherein (a) the patch comprisesa biosoluble & biodegradable matrix material and an influenza vaccine,and (b) the process comprises steps of: (i) dissolving the patch in asolvent to provide a dissolved patch solution; (ii) precipitatingproteins in the dissolved patch solution; and (iii) assayinghemagglutinin after precipitation in step (ii).

The Biodegradable Microneedles

Influenza vaccine is delivered via solid biodegradable microneedles.

The microneedles are solid, such that they retain their structuralintegrity during storage and can penetrate a subject's skin when thepatch is applied. The mechanical characteristics which are required forskirt penetration depend on the organism in question, but they willusually have sufficient strength to penetrate human skin. Materials forforming suitable solid needles are readily available and these can betested to determine appropriate concentrations eta for any particularneed.

The microneedles are biosoluble and biodegradable. Thus the solidmaterial dissolves in the skin after the patch is applied, in contrastto the coated metal microneedles used in references 2 & 3. Havingdissolved, the material will then be metabolised to give harmlessend-products. The timescale for dissolving after applying the patch canvary, but dissolving will typically commence immediately after applyingthe patch (e.g. within 10 seconds) and may continue for e.g. up to 1minute, 5 minutes, minutes, 20 minutes, 30 minutes, 1 hour, 5 hours, 10hours, or 24 hours, until the microneedle has fully dissolved. Materialswith suitable in vivo dissolving kinetics are readily available andthese can be varied and tested to determine appropriate concentrationsetc. for any desired dissolution profile.

Suitable matrix materials for forming the microneedles will typically bebiosoluble and biodegradable polymers, and these may comprise one ormore carbohydrates. For example, the material may comprise a cellulose,a dextrin, a dextran, a disaccharide, a chitosan, a chitin, etc., ormixtures thereof. Other GRAS materials may also be used.

Suitable celluloses include, but are not limited to, cellulose, sodiumcarboxymethyl cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, and hydroxypropyl methylcellulose. Suitable dextrins include,but are not limited to, maltodextrin, cyclodextrin, amylodextrin,icodextrin, yellow dextrin, and white dextrins. Suitable disaccharidesinclude, but are not limited to, sucrose, lactose, maltose, trehalose,turanose, and cellobiose.

Suitable mixtures for forming biosoluble and biodegradable microneedlesinclude, but are not limited to, mixtures of (i) dextrin and trehalose,(ii) sucrose and sodium carboxymethyl cellulose.

The microneedles can penetrate the skin. They should be long enough topenetrate through the epidermis to deliver material into the dermis(i.e. intradermal delivery), but are ideally not so long that they canpenetrate into or past the hypodermis. They will typically be 100-2500μm long e.g. between 1250-1750 μm long, or about 1500 μm. At the time ofdelivery the tip may penetrate the dermis, but the base of the needlemay remain in the epidermis.

The microneedles can have various shapes and geometries. They willtypically be tapered with a skin-facing point e.g. shaped as pyramids orcones. A tapered microneedle with a widest diameter of <500 μm istypical.

A single patch will typically include a plurality of microneedles e.g.≥10, ≥20, ≥30, ≥40, ≥50, ≥60, ≥70, ≥80, ≥90, ≥100, ≥200, ≥300, ≥400,≥50, ≥750, ≥1000 or more per patch. Where a patch includes a pluralityof microneedles, it may comprise a backing layer to which all of themicroneedles are attached. A unitary backing layer with ≥20 projectingmicroneedles is typical. Where a patch includes a plurality ofmicroneedles, these can be arranged in a regular repeating pattern orarray, or may be arranged irregularly.

A patch will typically have an area of 3 cm² or less, for example <2 cm²or <1 cm². A circular patch with a diameter of between 0.5 cm and 1.5 cmis useful.

The density of microneedles on a patch can vary, but may be ≥10 cm⁻²,≥20 cm⁻², ≥30 cm⁻², ≥40cm⁻², ≥50 cm⁻², ≥60 cm⁻², ≥70 cm⁻², ≥80 cm⁻² ormore.

A patch of the invention has a skin-facing inner face and anenvironment-facing outer face. The inner face may include an adhesive tofacilitate adherence to a subject's skin. When present, it is preferablynot present on the microneedles themselves i.e. the microneedles areadhesive-free. For example, a patch may have an additional backing whichprovides an outer adhesive margin for adhering the patch to skin e.g. asseen in sticking plasters or nicotine patches.

Patches as described above can be made by following the techniques andguidance in references 4-9.

For instance, a mold with 1.5 mm-long microneedle cavities can beprepared. A matrix material of dextrin and trehalose can be combinedwith an influenza vaccine and this aqueous material can be centrifugallycast in the mold to form an array of solid microneedles. A cellulose gelcan then be cast over the matrix/vaccine film to form a backing layer onthe patch. When this layer has dried, it can be removed to give a patchfrom which the solid microneedles project. Thus a process of theinvention may include, after step (ii), further steps of: (iii) lettingthe mixture set in the mold, to form solid microneedles; (iv)optionally, applying material to the set microneedles to provide abacking layer; and (v) removing the microneedles (and optional backinglayer) from the mold.

Patches of the invention may be packaged into individual pouches e.g.sealed under nitrogen, then heat sealed. They should be stored carefullyto avoid damage to the microneedles.

Surface Antigen Influenza Vaccines

Some embodiments of the invention use a surface antigen influenzavaccine. Such vaccines contain fewer viral components than a split orwhole virion vaccine. They include the surface antigens hemagglutininand, typically, also neuraminidase. Processes for preparing theseproteins in purified form from influenza viruses are well known in theart. The FLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are examples ofsurface antigen influenza vaccines.

The ability to administer surface antigen influenza vaccines using solidbiosoluble biodegradable microneedles is advantageous. Other intradermalneedle formats have been found to be incompatible with the high level ofresidual detergent that can be present in surface antigen influenzavaccines, but the solid biodegradable microneedle format is effectiveeven in these circumstances. Products of the invention may comprisedetergent (e.g. a non-ionic detergent) at between 0.05-50 μg per μg ofHA, e.g. as described in more detail below.

Where the invention uses a surface antigen influenza vaccine, this virusmay have been grown in eggs. The current standard method for influenzavirus growth for vaccines uses embryonated SPF hen eggs, with virusbeing purified from the egg contents (allantoic fluid). If egg-basedviral growth is used then one or more amino acids may be introduced intothe allantoid fluid of the egg together with the virus[16].

Virus is first grown in eggs. It is then harvested from the infectedeggs. Virions can be harvested from the allantoic fluid by variousmethods. For example, a purification process may involve zonalcentrifugation using a linear sucrose gradient solution that includesdetergent to disrupt the virions.

Antigens may then be purified, after optional dilution, bydiafiltration. Chemical means for inactivating a virus include treatmentwith an effective amount of one or more of the following agents:detergents, formaldehyde, β-propiolactone, methylene blue, psoralen,carboxyfullerene (C60), binary ethylamine, acetyl ethyleneimine, orcombinations thereof. Non-chemical methods of viral inactivation areknown in the art, such as for example DV light or gamma irradiation.

Other Forms of Influenza Vaccines

Some embodiments of the invention (specifically those which usecell-culture derived antigens, those which are not trivalent, thosewhich are mercury-free, and those which are gelatin-free) are notrestricted to using a surface antigen influenza vaccine. Theseembodiments may thus use whole inactivated virus, split virus,virosomes, live attenuated virus, or recombinant hemagglutinin. Thesevaccines can easily be distinguished from surface antigen vaccines bytesting their antigens e.g. for the presence of extra influenza virusproteins.

Whole inactivated virions can be obtained by harvesting virions fromvirus-containing fluids (e.g. obtained from eggs or from culture medium)and then treating them as described above.

Split virions are obtained by treating purified virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate,Triton X-100, Triton N-101, cetyltrimethylammonium bromide, TergitolNP9, etc.) to produce subvirion preparations, including the‘Tween-ether’ splitting process. Methods of splitting influenza viruses,for example are well known in the art e.g. see refs. 11-16, etc.Splitting or the virus is typically carried out by disrupting orfragmenting whole virus, whether infectious or non-infectious with adisrupting concentration of a splitting agent. The disruption results ina hill or partial solubilisation of the virus proteins, altering theintegrity of the virus. Preferred splitting agents are non-ionic andionic (e.g. cationic) surfactants e.g. alkylglycosides,alkylthioglycosides, acyl sugars, sulphobetaines, betains,polyoxyethylene-alkylethers, N,N-dialkyl-Glucamides, Hecarneg,alkylphenoxy-polyethoxyethanols, NP9, quaternary ammonium compounds,sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butylphosphate, myristyltrimethylammonium salts, lipofectin, lipofectarnine,and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols (e.g. the Tritonsurfactants, such as Triton X-100 or Triton N101), polyoxyethylenesorbitan esters (the Tween surfactants), polyoxyethylene ethers,polyoxyethlene esters, etc. One useful splitting procedure uses theconsecutive effects of sodium deoxycholate and formaldehyde, andsplitting can take place during initial virion purification (e.g. in asucrose density gradient solution). Thus a splitting process can involveclarification of the virion-containing material (to remove non-virionmaterial), concentration of the harvested virions (e.g. using anadsorption method, such as CaHPO₄ adsorption), separation of wholevirions from non-virion material, splitting of virions using a splittingagent in a density gradient centrifugation step (e.g. using a sucrosegradient that contains a splitting agent such as sodium deoxycholate),and then filtration (e.g. ultrafiltration) to remove undesiredmaterials, Split virions can usefully be resuspended in sodiumphosphate-buffered isotonic sodium chloride solution. Examples of splitvaccines are the BEGRIVAC™, INTANZA™, FLUARIX™, FLUZONE™ and FLUSHIELDT™products.

Virosomes are nucleic acid free viral-like liposomal particles [17].They can be prepared by solubilization of virus with a detergentfollowed by removal of the nucleocapsid and reconstitution of themembrane containing the viral glycoproteins. An alternative method forpreparing virosomes involves adding viral membrane glycoproteins toexcess amounts of phospholipids, to give liposomes with viral proteinsin their membrane.

Live attenuated viruses are obtained from viruses (grown in eggs or incell culture), but the viruses are not inactivated. Rather, the virus isattenuated (“att”) e.g. so as not to produce influenza-like illness in aferret model of human influenza infection. It may also be a cold-adapted(“ca”) strain i.e. it can replicate efficiently at 25° C., a temperaturethat is restrictive for replication of many wildtype influenza viruses.It may also be temperature-sensitive (“ts”) i.e. its replication isrestricted at temperatures at which many wild-type influenza virusesgrow efficiently (37-39° C.). The cumulative effect of the ca, ts, andalt phenotype is that the virus in the attenuated vaccine can replicatein the nasopharynx to induce protective immunity in a typical humanpatient, but it does not cause disease i.e. it is safe for generaladministration to the target human population. These viruses can beprepared by purifying virions from virion-containing fluids e.g. afterclarification of the fluids by centrifugation, then stabilization withbuffer (e.g. containing sucrose, potassium phosphate, and monosodiumglutamate). Live vaccines include the FLUMIST™ product. Although livevaccines can be used with the invention, it is preferred to use non-livevaccines.

As an alternative to using antigens obtained from virions,haemagglutinin can be expressed in a recombinant host (e.g. in an insectcell line, such as Sf9, using a baculovirus vector) and used in purifiedform [18-20] or in the form of virus-like particles (VLPs; e.g. seereferences 21 & 22).

Influenza Vaccines From Cell Culture

Some embodiments of the invention use influenza vaccine prepared fromviruses which were grown in cell culture, rather than in eggs.

When cell culture is used, the viral growth substrate will typically bea cell line of mammalian origin. Suitable mammalian cells of origininclude, but are not limited to, hamster, cattle, primate (includinghumans and monkeys) and dog cells. Various cell types may be used, suchas kidney cells, fibroblasts, retinal cells, lung cells, etc. Examplesof suitable hamster cells are the cell lines having the names BHK21 orHKCC. Suitable monkey cells are e.g. African green monkey cells, such askidney cells as in the Vero cell line. Suitable dog cells are e.g.kidney cells, as in the MDCK cell line. Thus suitable cell linesinclude, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5;PER.C6; W1-38; etc. Preferred mammalian cell lines for growing influenzaviruses include: MDCK. cells [23-26], derived from Madin Darby caninekidney; Vero cells [27-29], derived from African green monkey(Cercopithecus aethiops) kidney; or PER.C6 cells [30], derived fromhuman embryonic retinoblasts. These cell lines are widely available e.g.from the American Type Cell Culture (ATCC) collection, from the CoriellCell Repositories, or from the European Collection of Cell Cultures(ECACC). For example, the ATCC supplies various different Vero cellsunder catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and itsupplies MDCK cells under catalog number CCL-34. PER.C6 is availablefrom the ECACC under deposit number 96022940. As a less-preferredalternative to mammalian cell lines, virus can be grown on avian celllines [e.g. refs. 31-33], including cell lines derived from ducks (e.g.duck retina) or hens. Examples of avian cell lines include avianembryonic stem cells [31,34] and duck retina cells [32]. Suitable avianembryonic stern cells, include the EBx cell line derived from chickenembryonic stern cells, E845, EB14, and EB14-074 [35]. Chicken embryofibroblasts (CFI) may also he used.

The most preferred cell lines for growing influenza viruses are MDCKcell lines. The original MDCK cell line is available from the ATCC asCCL-34, but derivatives of this cell line may also be used. Forinstance, reference 23 discloses a MDCK cell line that was adapted forgrowth in suspension culture (‘MDCK 33016’, deposited as DSM ACC 2219).Similarly, reference 36 discloses a MDCK-derived cell line that grows insuspension in serum-free culture (‘B-702’, deposited as FERM BP-7449).Reference 37 discloses non-tumorigenic MDCK cells, including ‘MDCK-S’(ATCC PTA-6500), ‘MDCK-SE101’ (ATCC PTA-6501), ‘MDCK-SF102’ (ATCCPTA-6502) and ‘MDCK.-SF103’ (PTA-6503). Reference 38 discloses MDCK celllines with high susceptibility to infection, including ‘MDCK.5F1’ cells(ATCC CRL-12042). Any of these MDCK cell lines can be used.

Where virus has been grown on a mammalian cell line then products of theinvention will advantageously be free from egg proteins (e.g. ovalbuminand ovomucoid) and from chicken DNA, thereby reducing potentialallergenicity.

Hemagglutinin in cell-derived products of the invention can have adifferent glycosylation pattern from the patterns seen in egg-derivedviruses. Thus the HA (and other glycoproteins) may include glycoformsthat are not seen in chicken eggs. Useful HA includes canine glycoforms.

The absence of egg-derived materials and of chicken glycoforms providesa way in which vaccine prepared from viruses grown in cell culture canbe distinguished from egg-derived products.

Where virus has been grown on a cell line then the culture for growth,and also the viral inoculum used to start the culture, will preferablybe free from (i.e. will have been tested for and given a negative resultfor contamination by) herpes simplex virus, respiratory syncytial virus,parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus,reoviruses, potyomaviruses, bimaviruses, circoviruses, and/orparvoviruses [39]. Absence of herpes simplex viruses is particularlypreferred.

For growth on a cell line, such as on MDCK cells, virus may he grown oncells in suspension [23, 40, 41] or in adherent culture, One suitableMDCK. cell tine for suspension culture is MOCK 33016 (deposited as DSMACC 2219). As an alternative, microcarrier culture can be used.

Cell lines supporting influenza virus replication are preferably grownin serum-free culture media and/or protein free media. A medium isreferred to as a serum-free medium in the context of the presentinvention in which there are no additives from serum of human or animalorigin. Protein-free is understood to mean cultures in whichmultiplication of the cells occurs with exclusion of proteins, growthfactors, other protein additives and non-serum proteins, but canoptionally include proteins such as trypsin or other proteases that maybe necessary for viral growth. The cells growing in such culturesnaturally contain proteins themselves.

Cell lines supporting influenza virus replication are preferably grownbelow 37° C. during viral replication e.g. 30-36° C., at 31-35° C., orat 33+1° C.

The method for propagating virus in cultured cells generally includesthe steps of inoculating the cultured cells with the strain to becultured, cultivating the infected cells for a desired time period forvirus propagation, such as for example as determined by virus titer orantigen expression (e.g. between 24 and 168 hours after inoculation) andcollecting the propagated virus. The cultured cells are inoculated witha virus (measured by PFU or TCID₅₀) to cell ratio of 1:500 to 1:1,preferably 1:100 to 1:5, more preferably 1:50 to 1:10. The virus isadded to a suspension of the cells or is applied to a monolayer of thecells, and the virus is absorbed on the cells for at least 60 minutesbut usually less than 300 minutes, preferably between 90 and 240 minutesat 25° C. to 40° C., preferably 28° C. to 37° C. The infected cellculture (e.g. monolayers) may be removed either by freeze-thawing or byenzymatic action to increase the viral content of the harvested culturesupernatants. The harvested fluids are then either inactivated or storedfrozen. Cultured cells may be infected at a multiplicity of infection(“m.o.i.”) of about 0.0001 to 10, preferably 0.002 to 5, more preferablyto 0.001 to 2. Still more preferably, the cells are infected at a m.o.iof about 0.01. Infected cells may be harvested 30 to 60 hours postinfection. Preferably, the cells are harvested 34 to 48 hours postinfection. Still more preferably, the cells are harvested 38 to 40 hourspost infection, Proteases (typically trypsin) are generally added duringcell culture to allow viral release, and the proteases can be added atany suitable stage during the culture.

A vaccine product including vaccine prepared from cell culturepreferably contains less than 10 ng (preferably less than 1 ng, and morepreferably less than 100 pg) of residual host cell DNA per dose,although trace amounts of host cell DNA may be present.

it is preferred that the average length of any residual host cell DNA isless than 500 bp e.g. less than 400 bp, less than 300 bp, less than 200bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 43 & 44, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Treatment with an alkylatingagent, such as β-propiolactone, can also be used to remove host cellDNA, and advantageously may also be used to inactivate virions [45].

Influenza Vaccine Valency

Some embodiments of the invention use a monovalent influenza vaccine(i.e. it includes hemagglutinin antigen from a single influenza virusstrain) but in some embodiments it may be a multivalent vaccine, such asa trivalent vaccine, a tetravalent vaccine, or a >4-valent vaccine (i.e.including hemagglutinin from more than four different influenza virusstrains). Monovalent and multivalent vaccines are readily distinguishedby testing for multiple HA types, by amino acid sequencing, etc.

A monovalent vaccine is particularly useful for immunising against apandemic or potentially-pandemic strain, either during a pandemic or ina pre-pandemic situation. Characteristics of these strains are: (a) theycontain a new hemagglutinin compared to the hemagglutinins incurrently-circulating human strains, Le. one that has not been evidentin the human population for over a decade (e.g. H2), or has notpreviously been seen at all in the human population (e.g. H5, H6 or H9,that have generally been found only in bird populations), such that thehuman population will be immunologically naïve to the strain'shemagglutinin; (h) they are capable of being transmitted horizontally inthe human population; and (c) they are pathogenic to humans. Thesestrains may have any of influenza A HA subtypes H1, H2, H3, H4, H5, H6,H7, H8, H9, H10, H11, H12, H-13, H14, H15 or H16. A virus with H5hemagglutinin type is preferred for immunizing against pandemicinfluenza, or a H2, H7 or H9 subtype. The invention may protect againstone or more of influenza A virus NA subtypes Ni, N2, N3, N4, N5, N6, N7,N8 or N9. Thus possible strains include H5N1, H5N3, H9N2, H2N2, H7N1 andH7N7, and any other emerging potentially pandemic strains. In someembodiments, the invention does not use a monovalent vaccine based on aH1N1 strain e.g. it does not use mouse-adapted A/PR/8/34 H1N1 strain.

A multivalent vaccine is more typical in a seasonal setting e.g. atrivalent vaccine is typical, including hemagglutinins from twoinfluenza A virus strains and one influenza B virus strain, such as froma H1N1 influenza A strain, a H3N2 influenza A virus strain, and aninfluenza B virus strain. A tetravalent vaccine is also useful [46] e.g.including antigens from two influenza A virus strains and two influenzaB virus strains, or three influenza A virus strains and one influenza Bvirus strain. Thus a vaccine may be bivalent, trivalent, tetravalent,etc. Except for monovalent vaccines, it is usual to includehemagglutinin from both influenza A and influenza B virus strains. Invaccines including only two influenza A virus strains, these willusually be one H1 strain (e.g. a H1N1 strain) and one H3 strain (e.g. aH3N2 strain). In some embodiments, however, there may be one pandemicinfluenza A virus strain and one H1 strain, or one pandemic influenza Avirus strain and one H3 strain.

Where a vaccine includes more than one strain of influenza, thedifferent strains are typically grown separately and are mixed after theviruses have been harvested and antigens have been prepared. Thus aprocess of the invention may include the step of mixing antigens frommore than one influenza strain.

As described in reference 46, exemplary tetravalent vaccines can includehemagglutinin from two influenza A virus strains and two influenza Bvirus strains (‘A-A-B-B’), or from three influenza A virus strains andone influenza B virus strain (‘A-A-A-13’).

Influenza B virus currently does not display different HA subtypes, butinfluenza B virus strains do fall into two distinct lineages. Theselineages emerged in the late 1980s and have HAs which can be

antigenically and/or genetically distinguished from each other [47].Current influenza B virus strains are either B/Victoria/2/87-like orB/Yamagata/16/88-like. Where a vaccine of the invention includes twoinfluenza B strains, this will usually be one B/Victoria/2/87-likestrain and one B/Yamagata/16/88-like strain. These strains are usuallydistinguished antigenically, but differences in amino acid sequenceshave also been described for distinguishing the two lineages e.g.B/Yamagata/16/88-like strains often (but not always) have HA proteinswith deletions at amino acid residue 164, numbered relative to the‘Lee40’ HA sequence [48].

Preferred A-A-B-B vaccines include hemagglutinins from: (i) a H1N1strain; (iii) a H3N2 strain; (iii) a B/Victoria/2/87-like strain; and(iv) B/Yamagata/16/88-like strain.

In vaccines including three influenza A virus strains, these willusually be one H1 strain (e.g. a H1N1 strain) and two H3 strains (e.g.two H3N2 strains). The two H3 strains will have antigenically distinctHA proteins e.g. one H3N2 strain that cross-reacts with A/Moscow/10/99and one H3N2 strain that cross-reacts with A/Fujian/411/2002. The two H3strains may be from different clades (clades A, B and C of H3N2 strainsare disclosed in reference 49). In some embodiments, however, one ofthese strains (i.e. H1, or one of the two H3 strains) may be replaced bya pandemic strain.

Thus one preferred A-A-A-B vaccine includes hemagglutinins from: (1) aH1N1 strain; (ii) a A/Moscow/10/99-like H3N2 strain; (iii) aA/Fujian/411/2002-like H3N2 strain; and (iv) an influenza B virusstrain, which may be B/Victoria/2/87-like or B/Yamagata/16/88-like.

Another preferred A-A-A-B vaccine includes hemagglutinins from: (i) aH1N1 strain, (ii) a H3N2 strain, (iii) a H5 strain (e.g. a H5N1 strain)and (iv) an influenza B strain.

Another preferred A-A-A-B vaccine includes hernagglutinins from: (i) twodifferent H1 strains, (ii) a H3N2 strain, and (iii) an influenza Bstrain.

Where antigens are present from two or more influenza B virus strains,at least two of the influenza B virus strains may have distincthemagglutinins but related neuraminidases. For instance, they may bothhave a B/Victoria/2/87-like neuraminidase or may both have aB/Yamagata/16/88-like neuraminidase. For instance, twoB/Victoria/2/87-like neuraminidases may both have one or more of thefollowing sequence characteristics: (1) not a serine at residue 27, butpreferably a leucine; (2) not a glutamate at residue 44, hut preferablya lysine; (3) not a threonine at residue 46, but preferably anisoleueine; (4) not a proline at residue 51, but preferably a serine;(5) not an arginine at residue 65, but preferably a histidine; (6) not aglycine at residue 70, hut preferably a glutamate; (7) not a leucine atresidue 73, hut preferably a phenylalanine; and/or (8) not a proline atresidue 88, but preferably a glutamine. Similarly, in some embodimentsthe neuraminidase may have a deletion at residue 43, or it may have athreonine; a deletion at residue 43, arising from a trinucleotidedeletion in the NA gene, has been reported as a characteristic ofB/Victoria/2/87-like strains, although recent strains have regainedThr-43 [50]. Conversely, of course, the opposite characteristics may beshared by two B/Yamagata/16/88-like neuraminidases e.g. S27, E44, T46,P51, R65, G70, L73, and/or P88. These amino acids are numbered relativeto the ‘Lee40’ neuraminidase sequence [51]. Thus a A-A-B-B vaccine ofthe invention may use two B strains that are antigenically distinct forHA (one B/Yamagata/16/88-like, one B/Victoria12/87-like), but arerelated for NA (both B/Yamagata/16/88-like, or bothB/Victoria/2/87-like).

In some embodiments, the invention does not encompass a trivalent splitvaccine containing hemagglutinin from each of A/New Caledonia/20/99(H1N1), A/Wyoming/03/2003 (H3N2) and B/Jiangsu/10/2003 strains.

Strains whose antigens can usefully be included in the compositionsinclude strains which are resistant to antiviral therapy (e.g. resistantto oseltamivir and/or zanamivir), including resistant pandemic strains[53].

Vaccines Free From Certain Additives

The preparation of vaccines without the use of certain components andadditives is disclosed in reference 54, thereby ensuring that thesematerials are not present even in residual amounts.

In some embodiments of the invention, a vaccine may include a smallamount of mercury-based preservative, such as thiomersal or merthiolate.When present, such preservatives will typically provide less than Sag/mlmercury, and lower levels are possible e.g. <1 μg/ml, <0.5 μg/ml.Preferred vaccines are free from thiomersal, and are more preferablymercury-free [15,55]. Such vaccines may include a non-mercurialpreservative. Non-mercurial alternatives to thiomersal include2-phenoxyethanol or α-tocopherol succinate [15]. Most preferably, avaccine is preservative-free.

In some embodiments, a vaccine may include a stabilising amount ofgelatin e.g. at less than 0.1%. In other embodiments, however, a vaccineis gelatin-free. The absence of gelatin can assure that the vaccine issate in the small proportion of patients who are gelatin-sensitive[56,57].

In some embodiments, a vaccine may include one or more antibiotics e.g.neomycin, kanamycin, polymyxin B. In preferred embodiments, though, thevaccine is free from antibiotics.

In some embodiments, a vaccine may include formaldehyde. In preferredembodiments, though, the vaccine is free from formaldehyde.

As mentioned above, in some embodiments a vaccine may include eggcomponents (e.g. ovalbumin and ovomucoid), but preferred embodiments arefree from egg components.

Where a vaccine is described herein as being free from any particularcomponent, the same limitation is also disclosed in relation to patches,processes and materials of the invention.

Antigen Content

Hemagglutinin (HA) is the main immunogen in current inactivatedinfluenza vaccines, and vaccine doses are standardised by reference toHA levels, typically measured by STUD. Existing vaccines typicallycontain about 15 μg of HA per strain, although lower doses can be usede.g. for children, or in pandemic situations, or when using an adjuvant.Fractional doses such as ½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ havebeen used, as have higher doses (e.g. 3× or 9× doses [58,59]). Thesevaccines have a dosage volume or 0.5 ml i.e. a typical HA concentrationof 30 μg/ml/strain. The trivalent INTANZA™ product contains 9 μg of HAper strain in a 0.1 ml volume i.e. a HA concentration of 90μg/ml/strain, giving a total HA concentration of 270 μg/ml.

Products of the present invention can include between 0.1 and 50 μg ofHA per influenza strain per dose, preferably between 0.1 and 50 μg e.g.1-20 μg. Ideally a product has ≤16 μg hemagglutinin per strain e.g. 1-15μg, 1-10 μg, 1-7.5 μg, 1-5 μg, etc. Particular HA doses per straininclude e.g. about 15, about 10, about 7.5, about 5, about 3.8, about1.9, about 1.5, etc.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content e.g. a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) per strain per dose.

Influenza strains used with the invention may have a natural HA as foundin a wild-type virus, or a modified HA. For instance, it is known tomodify HA to remove determinants (e.g. hyper-basic regions around theHA1/HA2 cleavage site) that cause a virus to be highly pathogenic inavian species. The use of reverse genetics facilitates suchmodifications.

Vaccine Products

Vaccine products of the invention can include components in addition tothe biosoluble and biodegradable matrix material and influenza vaccineantigens.

As mentioned above, vaccine products may include a detergent. The levelof detergent can vary widely e.g. between 0.05-50 μg detergent per μg ofHA (‘μg/μg’). A low level of detergent can be used e.g. between 0.1-1μg/μg, or a high level can be used e.g. between 5-30 μg/μg. Thedetergent may he a single detergent (e.g. polysorbate 80, or CTAB) or amixture (e.g. both polysorbate 80 and CTAB). Preferred detergents arenon-ionic, such as polysorbate 80 (‘Tween 80’) or octyl phenolethoxylate (‘Triton X100’). Polysorbate 80 may be present at between0.05-50 μg polysorbate 80 per μg of HA e.g. between 0.1-1 μg/μg, 0.1-0.8μg/μg, 0.1-0.5 μg/μg, 5-40 μg/μg, 5-30 μg/μg, or 8-25 μg/μg.

As mentioned above, some vaccine products may include preservatives suchas thiomersal or 2-phenoxyethanol, but preferred vaccines are mercury-or preservative-free.

Vaccine products may include a physiological salt, such as a sodiumsalt. Sodium chloride (NaCl) is preferred, which may be present atbetween 1 and 20 mg/ml. Other salts that may be present includepotassium chloride, potassium dihydrogen phosphate, disodium phosphatedehydrate, magnesium chloride, calcium chloride, etc.

Vaccine products may include one or more buffers. Typical buffersinclude: a phosphate butler; a Tris butler; a borate buffer; a succinatebuffer; a histidine butler (particularly with an aluminum hydroxideadjuvant); or a citrate buffer. Buffers will typically be included inthe 5-20 mM range.

Vaccine products are preferably sterile. Vaccine products are preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. Vaccine products arepreferably gluten-free.

Vaccine products can include immunostimulatory molecules. These can bemixed with antigen before preparing a patch. Suitable classes ofimmunostimulatory molecule include, but are not limited to: TLR3agonists; TLR4 agonists; TLR5 agonists; TLR7 agonists; TLR8 agonists;TLR9 agonists; and CD1d agonists. Suitable immunostimulatory moleculesinclude, but are not limited to: imidazoquinolines such as imiquimod(“R-837”) [60,61] and resiquimod (“R-848”) [62], or salts thereof (e.g.the hydrochloride salts); aminoalkyl glucosaminide phosphatederivatives, such as RC-529 [63,64]; α-glycosylceramides, such asα-galactosylceramide; ‘ER 804057’ from reference 65; E5564 [66,67]; etc.

Methods of Treatment, and Administration of the Vaccine

Patches of the invention can be used to deliver an influenza vaccine toa subject via their skin. Thus the invention provides a method ofraising an immune response in a subject, comprising the step of applyinga patch of the invention to the subject's skin, such that the patch'smicroneedles penetrate the subject's dermis.

The invention also provides a patch of the invention for use in a methodof intradermal vaccination of a subject. The invention also provides theuse of (i) a biosoluble and biodegradable matrix material and (ii) aninfluenza vaccine selected from the group consisting of a purifiedinfluenza virus surface antigen vaccine, an influenza vaccine preparedfrom viruses grown in cell culture, and a monovalent influenza vaccine,in the manufacture of a medicament for raising an immune response in asubject.

Patches are suitable for administering vaccines to human or non-humananimal subjects

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses, neutralising capability andprotection after influenza virus vaccination are well known in the art.Human studies have shown that antibody titers against hen/agglutinin ofhuman influenza virus are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus) [68]. Antibodyresponses are typically measured by hemagglutination inhibition, bymicroneutralisation, by single radial immunodiffusion (STUD), and/or bysingle radial hemolysis (SRH). These assay techniques are well known inthe art.

Patches may be applied to the skin by simple manual application (e.g. aswith a sticking plaster or with known skin patches) or may be appliedusing a spring-driven injector.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Influenza vaccines are currently recommended foruse in pediatric and adult immunisation, from the age of 6 months. Thusa human subject may be less than 1 year old, 1-5 years old, 5-15 yearsold, 15-55 years old, or at least 35 years old. Preferred subjects forreceiving the vaccines are the elderly (e.g. ≥50 years old, ≥60 yearsold, and preferably ≥65 years), the young (e.g. ≤5 years old),hospitalised subjects, healthcare workers, armed service and militarypersonnel, pregnant women, the chronically ill, immunodeficientsubjects, subjects who have taken art antiviral compound (e.g. anoseltamivir or zanamivir compound; see below) in the 7 days prior toreceiving the vaccine, people with egg allergies and people travellingabroad. The vaccines are not suitable solely for these groups, however,and may be used more generally in a population, For pandemic strains,administration to all age groups is preferred.

Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMPcriteria for efficacy. In adults (18-60 years), these criteria are: (1)≥70% seroprotection; (2) 40% seroconversion; and/or (3) a GMT increaseof ≥2.5-fold. In elderly (>60 years), these criteria are: (1) ≥60%seroprotection; (2) ≥30% seroconversion; and/or (3) a GMT increase of≥2-fold. These criteria are based on open label studies with at least 50patients.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. Administration of more than one dose(typically two doses) is particularly useful in immunologically naïvepatients e.g. for people who have never received an influenza vaccinebefore, or for vaccinating against a new RA subtype (as in a pandemicoutbreak), Multiple doses will typically be administered at least 1 weekapart (e.g. about 2, weeks, about 3 weeks, about 4 weeks, about 6 weeks,about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

Assays

The invention also provides assays for determining the amount ofinfluenza hemagglutinin in a skin patch which comprises a biosoluble &biodegradable matrix material and an influenza vaccine. As shown below,the matrix materials do not interfere with an ELISA format and so thistechnique is suitable for analysing patches of the invention,particularly for quantitative analysis of HA content.

A patch is first dissolved in a suitable solvent (e.g. water or anaqueous buffer) to provide a dissolved patch solution. The dissolvedpatch solution is then assayed by ELISA, for example by a capture ELISAcomprising immobilised anti-hemagglutinin antibodies. If the patchcontains a multivalent influenza vaccine then the process may involveseparate assays for each valence e.g. by using strain-specific captureantibodies, one per strain.

After a patch is dissolved in a solvent the dissolved patch solution canbe treated to precipitate soluble proteins e.g. by addingtrichloroacetic acid (TCA), deoxycholate (DOC), acetone, methanol,chloroform, or mixtures thereof After precipitation the proteins can beassayed; some analytical methods may first require the proteins to bere-solubilised, As shown below, precipitation in this manner canincrease the recovery of protein for some analytical purposes.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±5%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

BRIEF DESCRIPTION THE DRAWINGS

FIG. 1 shows scanning electron micrograph images of a patch of theinvention, Panels B, C & D show individual needles from the patch shownin panel A.

FIG. 2 shows SDS-PAGE analysis of antigens, either in solution or afterformulation into a patch. Lanes are: (1) markers; (2) 3-valent antigenat 30 μg HA per strain; (3) 3-valent antigen at 15 μg HA/strain; (4)3-valent antigen at 7.5 μg HA/strain; (5-7) monovalent HAs at 15 μg; (8)empty patch after TCA treatment; (9) patch after TCA treatment.

FIG. 3 shows ELISA results for antigen from two different strains. Thecircles show data for a trivalent vaccine. The triangles show data witha dummy patch spiked with trivalent vaccine. The squares show data for apatch with integral trivalent vaccine. The crosses show a dummy patch.

FIG. 4 shows strain-specific IgG titers after immunisations. Each of theseven triplets of bars shows titers for the three strains in thetrivalent vaccine. The triplets are, from left to right: unadjuvantedinjected vaccine at 0.1 μg dose; patch-administered vaccine at 0.1 μgdose; adjuvanted injected vaccine at 0.1 μg dose; unadjuvanted injectedvaccine at 0.01 μg dose; patch-administered vaccine at 0.01 μg dose;adjuvanted injected vaccine at 0.01 μg dose; naïve mice.

FIG. 5 shows serum H1 titers. The bars are grouped as in FIG. 4 .

FIG. 6 shows strain-specific IgG titers. The five pairs of bars showtiters after 1 dose or 2 doses. The pairs are, from left to right:unadjuvanted injected vaccine at 0.1 μg dose; patch-administered vaccineat 0.1 μg dose; unadjuvanted injected vaccine at 1 μg dose;patch-administered vaccine at 1 μg dose; mice receiving PBS alone.

FIG. 7 shows serum H1 titers against one vaccine strain. The five groupsare as in FIG. 6 .

FIG. 8 shows % weight loss in mice after challenge. Diamonds show datafor unadjuvanted injected vaccine at 0.1 μg (empty) or 1 μg (filled).Squares show data for patch-administered vaccine at 0.1 μg (empty) or 1μg (filled). Crosses show data for mice receiving PBS alone.

FIG. 9 shows microneutralization titers (IC80). The five groups are asin FIG. 6 .

MODES FOR CARRYING OUT THE INVENTION Vaccine Patch Fabrication

An influenza virus vaccine was prepared using the MDCK cell culture andantigen purification techniques used for manufacturing the OPTAFLU™product [69]. This provides a surface antigen inactivated vaccine freefrom mercury, antibiotics, formaldehyde, and egg-derived materials.

Bulk monovalent antigens from each of A/H1N1, A/H3N2 and B strainsincluded a high HA concentration (200-600 μg/ml) with about 0.5% w/vTween 80. These three bulks were mixed to give a trivalent bulk at highHA concentration. This hulk was mixed with trehalose and sodiumcarboxymethylcellulose, and a microneedle patch was prepared by fillinga micromold with the mixture then centrifuging at 4000 rpm for 5minutes. The centrifuged material was then dried to give the patch.Antigens were incorporated to give a final concentration per patch of0.01 μg, 0.1 μg, 1 μg or 15 μg of HA per strain.

FIG. 1 shows scanning electron micrographs of a patch after sputtercoating with gold palladium alloy for two minutes.

Assays for Antigen in Fabricated Patches

To confirm that vaccine antigens were properly incorporated and stable,patches were characterized qualitatively by SDS-PAGE and quantitativelyby capture ELISA.

Patches containing trivalent antigen at 15 μg per strain were dissolvedin 1 ml sterile water. Vials were vortexed for 10 minutes to ensure theentire patch was in solution. 100 μl of 0.5% deoxycholate was added tothe samples. Samples were allowed to sit at room temperature for 10minutes. After incubation 80 μl of 60% TCA was added to the sample.Samples were placed on microcentrifuge for minutes at room temperatureat 12k RPM. The supernatant was removed and the pellet was dried. 60 μlof 4× reducing loading buffer and 20 μl of 1M Tris-HCl pH 8 was added tothe pellet. The sample was vortexed and placed on a heating block set at90° C. for 10 minutes. Samples were allowed to cool to room temperatureand were 9 μl was added to each well in a 4-20% SDS-PAGE gel. Gels werestained overnight, de-stained in distilled water, and imaged. Anantigen-free patch was treated in the same way for comparison.

FIG. 2 shows results. Lanes 2-4 contain non-patch trivalent antigen inlanes 2-4 at 2×, 1× and 0.5× the concentration in the patch. Lanes 5-7show non-patch monovalent antigens. Lane 8 shows an antigen-free patch,and lane 9 shows the TCA-precipitated patch. The three individualantigens are clearly visible in the patch.

Antigen content of the patches was analyzed by capture ELISA. In thistechnique ELISA plates were coated to capture the antigen. The dissolvedpatches were added to the plates and incubated, followed by biotinylatedIgG antibody for 30 min. Subsequently, unbound IgG and antigen waswashed off and a streptavidin antibody conjugated to alkalinephosphatase was added. Antigen content was then determined by enzymaticreaction with a pNPP substrate. Absorbance was measured at 405 nm andantigen concentration was extrapolated from antigen-specific standardcurves.

Results are shown in FIG. 3 . The capture ELISA was able to recover thefull antigen content from patches, confirming that the matrix excipientsfrom the patch do not interfere with the assay.

In contrast, mass spectrometry methods were able to recover around 50%of the HA content. Recovery was calculated by comparing the area of thepeak in the patch sample with the area of the peak in a standard mixsample, repeated with five different peptides for each strain. Thisprocess was performed on patches which had been treated with or withoutTCA to precipitate their proteins. Recovery for one strain was 17%without TCA or 43% with TCA; for another strain it was 24% without TCAor 49% with TCA. Spiking studies were also used, and recovery was againpoor (ranging from 41-55% across three different strains). Thus massspectrometry was not useful for quantifying HA in the patches,presumably due to some interference from the patch excipients.

Immunization and Challenge Studies

Patches for immunization studies had a much lower antigen content (1,0.1 or 0.01 μg HA per strain than the patches which were used forantigen assays (15 μg per strain).

In a first series of experiments patches were loaded at 0.1 or 0.01 μgHA per strain per patch. Patches were applied to shaved mice (femaleBalb/C mice, 8-10 weeks old) with pressure for 3 minutes, and thenremoved 15 minutes later, by which time the tips of the needles werecompletely dissolved. Two immunizations were carried out 30 days apartand serum samples were collected before the first immunization and twoweeks after each immunization. Individual serum samples were analyzedfor IgG titers by ELISA (FIG. 4 ) and hemagglutination inhibition (HI)titers (FIG. 5 ). The results of the ELISA indicate comparable IgGtiters upon intramuscular injection of trivalent influenza vaccine orupon patch administration at the 0.1 μg dose.

In a second series of experiments patches were loaded at 0.1 or 1 μg HAper strain per dose. Mice were immunised and assayed in the same way asbefore. FIG. 6 shows strain-specific IgG titers, and FIG. 7 shows H1results. In addition to these assays, two weeks after the secondimmunization the animals were challenged with one of the wild-typevaccine strains at 10 MLD₅₀ (300,000 TCID₅₀/mice). Animals weremonitored every two days for weight loss after challenge, and after 14days neutralization titers were determined to confirm protection.

FIG. 8 shows body weight. About 10-15% weight loss was observed in thefirst three days after viral challenge, but mice in the treated groupsrecovered within a week. In contrast, untreated control group suffered a˜20% weight loss and recovered only to 97% of original weight after twoweeks.

FIG. 9 shows neutralization titers, calculated as the sera dilution atwhich 80% of the cells are protected against virus infection. The titeris expressed as IC80 and calculated using a 4 parameter curve fitting.Administration of the vaccine via the patch at 0.1 μg dose resulted inneutralization titers slightly lower than non-adjuvanted vaccineadministered intramuscularly.

In conclusion, intradermal administration of influenza vaccine by thepatch induced HI titers for all three influenza strains which werecomparable to those achieved by intramuscular administration ofnon-adjuvanted vaccine. This effect was seen with HA doses as low as 0.1μg/strain. Additionally, ELISA results indicated comparable IgG titers.In the challenge study, both microneedle patches and non-adjuvantedinfluenza antigen at 0.1 and 1 μg doses resulted in positiveneutralization titers.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

[1] Vaccines. (eds. Plotkin & Orenstein). 4th edition. 2004, ISBN:0-7216-9688-0.

[2] Koutsonanos et al. (2009) PLoS ONE 4(e): e4773.

[3] Quan et at. (2009) PLoS ONE 4(9):e7152.

[4] WO2007/030477.

[5] U.S. Pat. No. 6,945,952.

[6] U.S. Pat. No. 7,211,062.

[7] Sullivan et al. (2008) Adv Mater 20:933-8.

[8] U.S. Pat. No. 7,182,747.

[9] Oh et al. (2006) American Association of Pharmaceutical Scientists,2006 Annual Meeting and Exposition. The AAPS Journal. 8(S2). (AnnualMeeting).

[10] Intradermal Delivery of Vaccines: A review of the literature andthe potential for development for use in low- and middle-incomecountries. (2009) Program for Appropriate Technology in Health.

[11] WO02/28422.

[12] WO02/067983.

[13] WO02/074336.

[14] WO01/21151.

[15] WO02/097072.

[16] WO2005/113756.

[17] Huckriede et al. (2003) Methods Enzymol 373:74-91.

[18] WO96/37624.

[19] WO98/46262.

[20] WO95/18861.

[21] Bright et at. (2008) PLoS ONE 3:e1501.

[22] Crevar & Ross (2008) Virology Journal 5:131.

[23] WO97/37000.

[24] Brands et al. (1999) Dev Biol. Stand 98:93-100.

[25] Halperin et al. (2002) Vaccine 20:1240-7.

[26] Tree et al. (2001) Vaccine 19:3444-50.

[27] Kistner et al. (1998) Vaccine 16:960-8.

[28] Kistner et al. (1999) Dev Biol Stand 98:101-110.

[29] Brutal et al. (2000) Vaccine 19:1149-58.

[30] Pau et al. (2001) Vaccine 19:2716-21.

[31] WO03/076601.

[32] WO2005/042728.

[33] WO03/043415.

[34] WO01/85938.

[35] WO2006/108846.

[36] EP-A-1260581 (WO01/64846).

[37] WO2006/071563.

[38] WO2005/113758.

[39] WO2006/027698.

[40] WO03/023021

[41] WO03/023025

[42] WO97/37001.

[43] EP-B-0870508.

[44] U.S. Pat. No. 5,948,410.

[45] WO2007/052163.

[46] WO2008/068631.

[47] Rota et al. (1992) J Gen Viral 73:2737-42.

[48] GenBank sequence G1:325176.

[49] Holmes et al. (2005) PLoS Biol. 3(9):e300.

[50] McCullers et al. (1999) J Virol 73:7343-8.

[51] GenBank sequence G1:325237.

[52] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.

[53] Le et al. (2005) Nature 437(7062):1108.

[54] WO2009/001217

[55] Banzhoff (2000) Immunology Letters 71:91-96.

[56] Lasley (2007) Pediatric Asthma Allergy & Immunology 20(3): 201-5.

[57] Coop et al. (2008) Int Arch Allergy Immunol. 146(1):85-8.

[58] Treanor et al. (1996) J Infect Dis 173:1467-70.

[59] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10.

[60] U.S. Pat. No. 4,680,338.

[61] U.S. Pat. No. 4,988,815.

[62] WO92/15582.

[63] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.

[64] Evans et al. (2003) Expert Rev Vaccines 2:219-229.

[65] WO03/011223.

[66] Wong et al. (2003) J Clin Pharmacol 43(7):735-42.

[67] US2005/0215517.

[68] Potter & Oxford (1979) Br Med Bull 35: 69-75.

[69] Doroshenko & Halperin (2009) Expert Rev Vaccines 8:679-88.

1. A skin patch comprising a plurality of solid biodegradablemicroneedles, wherein the microneedles comprise a mixture of (i) abiosoluble and biodegradable matrix material and (ii) influenza vaccineselected from the group consisting of a purified influenza virus surfaceantigen vaccine, art influenza vaccine prepared from viruses grown incell culture, a monovalent influenza vaccine, a bivalent vaccine, atetravalent or >4-valent vaccine, a mercury-free vaccine, and agelatin-free vaccine.
 2. A process for preparing a skin patch comprisinga plurality of solid biodegradable microneedles, comprising steps of:(i) mixing a biosoluble and biodegradable matrix material with aninfluenza vaccine selected from the group consisting of a purifiedinfluenza virus surface antigen vaccine, art influenza vaccine preparedfrom viruses grown in cell culture, a monovalent influenza vaccine, abivalent vaccine, a tetravalent or >-4-valent vaccine, a mercury-freevaccine, and a gelatin-free vaccine; and (ii) adding the mixture fromstep (i) to a mold containing cavities for forming microneedles.
 3. Anaqueous liquid or solid material comprising (i) a biosoluble andbiodegradable matrix material and (ii) an influenza vaccine selectedfrom the group consisting of a purified influenza. virus surface antigenvaccine, an influenza vaccine prepared from viruses grown in cellculture, a monovalent influenza vaccine, a bivalent vaccine, atetravalent or >4-valent vaccine, a mercury-free vaccine, and agelatin-free vaccine.
 4. The patch, process or material of any one ofclaims 1-3, wherein the influenza vaccine is a purified influenza virussurface antigen vaccine.
 5. A skin patch comprising a plurality of solidbiodegradable microneedles, wherein the microneedles comprise a mixtureof (i) a biosoluble and biodegradable matrix material and (ii) aninfluenza virus hemagglutinin, wherein the amount of influenza virushemagglutinin per patch is ≤16 μg per strain.
 6. The patch of claim 5,wherein the patch comprises a whole virus inactivated influenza vaccine,a split virus influenza vaccine, or a purified influenza virus surfaceantigen vaccine.
 7. The patch, process or material of any one of claims1-6, wherein the influenza vaccine is an influenza vaccine prepared fromviruses grown in cell culture.
 8. The patch, process or material of anyone of claims 1-6, wherein the influenza vaccine is a monovalentinfluenza vaccine.
 9. The patch, process or material of any one ofclaims 1-6, wherein the influenza vaccine is a bivalent influenzavaccine.
 10. The patch, process or material of any one of claims 1-6,wherein the influenza vaccine is a tetravalent influenza vaccine. 11.The patch, process or material of any one of claims 1-6, wherein theinfluenza vaccine is a >4-valent influenza vaccine.
 12. The patch,process or material of any one of claims 1-6, wherein the influenzavaccine is a mercury-free influenza vaccine.
 13. The patch, process ormaterial of any one of claims 1-6, wherein the influenza vaccine is agelatin-free influenza vaccine.
 14. The patch, process or material ofany preceding claim, wherein the matrix material comprises one or morecarbohydrates.
 15. The patch, process or material of claim 14, whereinthe matrix material comprises a cellulose and/or a dextrin and/or adisaccharide.
 16. The patch or process of any preceding claim, whereinthe microneedles are 100-2500 μm long and are tapered with a skin-facingpoint.
 17. The patch or process of any preceding claim, wherein a singlepatch has >20 microneedles.
 18. The patch or process of any precedingclaim, wherein the patch has an area of ≤2 cm².
 19. The patch or processof any preceding claim, wherein a skin-facing area of the patch includesan adhesive to facilitate adherence to a subject's skin.
 20. The patch,process or material of any preceding claim, comprising between 0.5-50 μgor detergent per μg of hemagglutinin.
 21. The patch, process or materialof any preceding claim, containing 1-15 μg, of hemagglutinin perinfluenza virus strain.
 22. A method of raising an immune response in asubject, comprising the step of applying a patch of any preceding claimto the subject's skin, such that the patch's microneedles penetrate theskin's dermis.
 23. A process for determining the amount of influenzahemagglutinin in a skin patch, wherein (a) the patch comprises abiosoluble & biodegradable matrix material and an influenza vaccine, and(b) the process comprises steps of (i) dissolving the patch in a solventto provide a dissolved patch solution; and (ii) assaying hemagglutininin the dissolved patch solution by enzyme-linked immunosorbent assay(ELISA).
 24. The process of claim 23, wherein the ELISA is a captureELISA using immobilised anti-hemagglutinin antibodies.
 25. The processof claim 23 or claim 24, wherein the influenza vaccine is a multivalentinfluenza vaccine, and wherein the ELISA separately uses strain-specificanti-hernagglutinin antibody for each strain.